Herpes zinc finger motifs

ABSTRACT

The present invention relates to a method for detecting an agent for use in the treatment of herpes virus infection and use of known agents, such as 2,2′-dithiobisbenzamide (DIBA) and azodicarbonamide (ADA), and unknown agents, which selectively eject zinc bound to a zinc finger protein, for the manufacture of a medicament for the treatment of herpesvirus infections.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 371 from PCIApplication No. PCT/GB01/03114, filed in English on Jul. 11, 2001, whichclaims the benefit of Great Britain Application Serial No. 0016890.6filed on Jul. 11, 2000, the disclosures of which are incorporated byreference herein in their entireties.

The present invention relates to a method for detecting an agent for usein the treatment of herpes virus infection and use of known and unknownagents for the manufacture of a medicament for the treatment ofherpesvirus infections.

A paradigm for the development of antivirals was based on theidentification of agents that selectively eject zinc from retroviralzinc finger proteins. One such protein is the human immunodeficiencyHIV-1 nucleocapsid protein NCp7, which is essential for early and latevirus replication. NCp7 interacts with the viral RNA at a packaging site(1). This 55 bp amino acid protein contains two zinc fingers, one ofwhich is highly conserved among the retroviruses (reviewed in 2). TheNCp7 CCHC zinc finger represents a rare conserved feature, absent incellular proteins, against the extreme variation of other retroviruscomponents which suggests that mutation to resistance against reagentsthat target conserved zinc finger motifs may be difficult to achieve.Potent anti-HIV-1 agents that selectively target the NCp7 protein zincfinger by ejecting zinc have been identified which are non-toxic tocells (3,4).

The following summarised documents describe the use and action of suchagents in the treatment of retroviral infections:

Huang et al. 1998 describe agents that target retroviral nucleocapsidprotein zinc fingers without seriously affecting cellular zinc fingerproteins. Specifically, the agents are 3-nitrosobenzamide (NOBA),disulfide benzamides (DIBAs or 2,2′-dithiobisbenzamides),dithiaheterocyclic molecules such as 1,2-dithiane-4,5-diol, 1,1-dioxide,cis(dithiane), α-carbonyl azoic compounds such as azodicarbonamide(ADA), and others. However, this document only discloses the agents'action on retroviruses and in particular nucleocapsid p7 (NCp7).

Vandervelde et al 1996 describe the anti-HIV-1 properties andclinical/pre-clinical data of 1,1′-azobisformamide (ADA), indicatingtoxicity studies of the compound in patients.

U.S. Pat. No. 5,516,941 describes c-nitroso compounds which destabilisezinc fingers such as 6-nitroso 1,2-benzopyrone (NOBP),2-nitrbsobenzamide, 3-nitrosobenzamide (NOBA), 4-nitrosobenzamide,5-nitroso-1(2H)-isoquinolinone (5-NOQ), 7-nitroso-1(2H)isoquinolin(5-NOQ), 8-nitroso-1(2H)-isoquinolinone (8-NOQ), and related compoundsincluding nicotinamides, pthalhydraziides and 1,3-benzoxazine-2,4 dionesfor inactivating retroviruses either alone or in combinations thereof.It also provides for methods of detecting compounds that can inactivateretroviruses by testing for the effect of zinc finger destabilising i.e.ejection of zinc measured using NMR.

U.S. Pat. No. 5,463,122 and its divisional application U.S. Pat No.5,668,178 describe the preparation and use of phenylthiols anddithiobisbenzamides in the treatment of retrovirus infections includingHIV treatment.

U.S. Pat No. 5,585,367 describe the use of azoic compounds (includingADA) in the treatment of retroviral infections. It also details clinicalstudies on the use of ADA but at no time does the article refer to thedrug's use in the treatment of viruses other than retroviruses.

In summary, although some of these documents detail the use and actionof specific drugs in the treatment of retroviral infection at no time isit inferred that such drugs can be used in the treatment ofnon-retrovirus infections such as herpesvirus infections.

Herpesviruses are one of the most important virus families and cause arange of prominent medical or veterinary diseases. Eight differentherpesviruses whose natural host is man have been identified so far:herpes simplex virus (HSV) types 1 and 2, varicella-zoster virus (VZV),Epstein-Barr virus, human cytomegalovirus (HCMV) and human herpesvirus(HHV) types 6-8 (reviewed in 5). A large proportion of people world-widehave been exposed to, and may be latently infected with, one or moreherpesviruses. Whereas in general an intact immune system keeps theseviruses in check, immunocompromised individuals are particularly atrisk. HCMV disease in graft recipients, newborns and immunocompromisedpatients accounts for considerable morbidity. VZV can be a severe andlife threatening problem for leukaemic children and patients undergoingchemotherapy or immunosuppressive drug treatment for organ transplants.Herpesviruses have been implicated in the aetiology of different typesof cancer with the recently discovered gammaherpesvirus HHV-8 implicatedin Kaposi's sarcoma, a major neoplasm of AIDS patients.

At present there are few antiviral agents of broad specificity againstthe herpesvirus family of viruses (6), and resistance to antiherpesvirusdrugs such as acyclovir, against HSV and VZV, and gancyclovir, againstHCMV, is an increasing problem (7,8,9), prompting the call for newtherapeutic approaches to herpesvirus infections (7).

It is an object of the present invention to obviate and/or mitigate atleast some of the above disadvantages.

Broadly speaking, the present invention is based on the presentinventors' observation that the spacing and metal co-ordinating residuesin the IE63 zinc finger of herpes simplex virus type I are conserved inall related homologues within the alphaherpesvirus sub-family. Similarconservation of spacing of zinc finger motifs but with differentarrangements of the conserved motif residues was also discovered by theinventors within the betaherpesvirus and gammaherpesvirus family.

IE63 (also called ICP27) is an essential HSV-1 protein (reviewed in 10).An IE63 homologue exists in all examples of mammalian and avianherpesviruses sequenced so far (around twenty). IE63 is the only HSV-1immediate early regulatory protein conserved in other herpesvirusindicative of its central role in viral gene regulation. The product ofthe HSV-1 and HSV-2 UL54 gene, IE63 (ICP27) has counterparts in otherherpesviruses namely VZV ORF 4, HCMV UL69, Epstein-Barr virus BMLF1(SM/MTA), HHV-6 U42, HHV-7 U42, HHV-8 ORF57, equine herpesvirus-1 ORF3,equine herpesvirus-4 ORF3, bovine herpesvirus-1 BICP27, pseudorabiesvirus UL54, Marek's disease virus UL54, murine cytomegalovirus M69,herpesvirus saimiri ORF57, bovine herpesvirus-4 ORF57, avian herpesvirus-1 ORF57 and murine herpesvirus-68 ORF57. HSV-1 IE63 is anessential immediate-early phosphoprotein for the progression of thevirus lytic replication cycle from immediate early to early and latestages, its functions affect the transcription of virus genes into RNAand subsequent post-transcriptional processing of this RNA. The actionof IE63 is complex, binding other herpesvirus proteins, colocalisingwith splicing factors and binding RNA with a preference for viralintron-less transcripts (11-13) and inhibiting splicing of both viraland cellular transcripts (14). IE63 causes the nuclear retention ofintron-containing transcripts while facilitating the nuclear export ofviral intron-less transcripts allowing the virus to take control of hostgene expression. The post-transcriptional action of IE63 is even morecomplex, enhancing 3′RNA processing at late viral poly(A) sites andstabilising 3′ends of mRNA.

IE63 is a zinc finger protein. The yeast genome encodes some 500 zincfinger proteins (reviewed in 11, 12) and estimates are that some 1% ofall mammalian genes encode such motifs. The action of zinc fingersinclude binding to DNA, RNA and DNA-RNA hybrids as well as mediatingprotein-protein interactions. potentiating self interaction andinteractions with other proteins. The IE63 zinc binding region itself(18) is required for self interaction (19, 20) and for interaction withCK2 (19).

The discovery by the present inventors that the spacing and metalco-ordinating residues of the IE63 zinc finger and its homologues areconserved within each of the herpesvirus sub-families (alpha-, beta- andgammaherpesvirus; Example 1)is analogous to the retrovirus HIV-1 proteinNCp7, which has been targeted for the development of antivirals thatselectively eject zinc (reviewed in 2).

Inspection of the databases reveals that the three conserved zinc fingermotifs within the alpha-, beta- and gammaherpesvirus families aredivergent to any other identified zinc finger motif present in viral andcellular proteins. Thus, like HIV-1 Ncp7, the IE63 zinc fingerrepresents a rare conserved feature, absent in cellular proteins,against the extreme variation of other virus, in this case herpesvirus,components. This discovery allows the development of new anti-herpesvirus agents.

Thus, in a first aspect of the present invention there is provided amethod for detecting an agent for use in the treatment of herpes virusinfection comprising the steps of:

-   (a) forming a herpes virus polypeptide/zinc complex;-   (b) adding a test agent to said polypeptide/zinc complex; and-   (c) detecting any change in the polypeptide/zinc complex.

The method is particularly suited for the detection of agents which mayserve as antiherpesvirus agents. The term antiherpesvirus agent isunderstood to mean an agent which when in contact with a herpesvirusand/or herpesvirus infected cells selectively kills and/or destabilisesviruses from this virus family only. Killing or destabilising ofherpesviruses may be achieved by, for example, interference of viralnucleic acid synthesis and/or regulation, virus-cell binding, virusuncoating or viral replication. Preferably, the action of theantiherpesvirus agent will not interfere unduly with the infected hostcells' machinery.

The term agent is understood to include chemicals, nucleic acidanalogues, peptides and/or proteins. For example, these may includeC-nitroso and related compounds or disulphide benzamides and azoiccompounds such as 2,2′-dithiobisbenzamide (DIBA) and azodicarbonamide(ADA) respectively.

According to the present invention, the term polypeptide refers toherpesvirus HSV-1 and ESV-2 IE63 protein, and counterparts in otherherpesviruses namely VZV ORF4, HCMV UL69, Epstein-Barr virus BMLF1(SM/MTA), HHV-6 U42, HHV-7 U42, HHV-8 ORF57, equine herpesvirus-1 ORF3,equine herpesvirus-4 ORF3, bovine herpesvirus-1 BICP27, pseudorabiesvirus UL54, Marek's disease virus UL54, murine cytomegalovirus M69,herpesvirus saimiri ORF57, bovine herpesvirus-4 ORF57, avian herpesvirus-1 ORF57 and murine herpesvirus-68 ORF57 as shown in FIG. 1, orfunctional derivatives or functional homologues thereof. In particular,it refers to a polypeptide comprising at least the C-terminal regions ofIE63 and homologues as shown in FIG. 2. More particularly, it refers topolypeptides comprising the zinc finger motifs of IE63 homologues of thealpha-, beta- or gammaherpesvirus family as highlighted in bold in FIG.2.

The term functional derivative thereof according to the presentinvention refers to any polypeptide containing at least the zinc fingermotif of herpesvirus IE63 or its counterparts as shown in FIGS. 1 and 2.The skilled addressee will appreciate that it is possible to manipulatea full-length protein in order to express derivatives of the full-lengthpolypeptide.

Furthermore, the term functional homologue refers to a polypeptide witha sequence similar to those depicted in FIGS. 1 and 2 with a conservedfunction.

It should be understood that the degree of similarity, over thefull-length polypeptide, of functional homologues may vary greatly. Thatis, a functional homologue is defined by the conservation of amino acidresidues in a motif required to characterise a protein family. Thus,there may be a low percentage of similarity of amino acid residuesoutwith a motif of interest in a full-length polypeptide but as long assaid motif is conserved the protein maintains its function and is thusregarded as a functional homologue. In terms of the present invention,it can be seen from FIGS. 1 and 2 that a high percentage of similarityof amino acid residues in herpesvirus IE63 and its homologues isconfined to the zinc-finger motif and there is a relative lack ofconservation outwith this motif. In particular, functional honologues ofIE63, according to the present invention are defined by the preciseconservation of spacing and metal co-ordinating amino acid residues inthe Zn-finger motif in each of the herpesvirus families. In more detail,Cys-X₁₀-His-X₃-Cys-X₄-Cys-X₁₄-His-X₆-Cys in the alphaherpesvirusZn-finger motif, Cys-X₁₂-His-X₁-Cys-X₄-Cys-X₁₇-Cys in thebetaherpesvirus Zn-finger motif and His-X₃-Cys-X₄-Cys in thegammaherpesvirus subfamily Zn-finger motif of IE63 functionalhomologues.

In a further aspect of the present invention there is provided a methodfor detecting an agent for use in the treatment of herpes virusinfection comprising the steps of:

-   a) forming a herpes virus polypeptide/zinc complex wherein said    polypeptide comprises at least the    Cys-X₁₀-His-X₃-Cys-X₄-Cys-X₁₄-His-X₆-Cys alphaherpesvirus subfamily    zinc-finger motif, or Cys-X₁₂-His-X₁-Cys-X₄-Cys-X₁₇-Cys    betaherpesvirus subfamily zinc-finger motif or His-X₃-Cys-X₄-Cys    gammaherpesvirus subfamily zinc finger motif of herpesvirus IE63    functional homologues;-   b) adding a test agent to said polypeptide/zinc complex; and-   c) detecting any change in the polypeptide/zinc complex.

In order to detect antiherpesvirus agents according to the presentinvention said polypeptide must first be complexed with zinc. Zinc maybe provided in the form of a standard dialysis buffer comprising a knownconcentration of zinc.

Detection of said antiherpesvirus is based on a change in the propertiesof said polypeptide/zinc complex when a test agent is added. Said changewill be a measure of the test agent's ability to eject zinc anddestabilise viral zinc fingers. For example, displacement of zinc fromsaid polypeptide/zinc complex is thus a measure of an agent's potentialas an antiherpesvirus agent. It should be understood that the propertiesof said polypeptide/zinc complex such as concentration and zinc contentof (purified) polypeptides may be measured by standard techniques suchas standard amino acid analysis and atomic adsorption spectroscopy.

Detection of a change in said polypeptide/zinc complex may be achievedamong other methods by using ⁶⁵Zn²⁺ or NMR, which can detect ejection ofzinc from said polypeptide/zinc complex by a test agent. For example,for the NCp7 zinc finger domain, loss of NMR signals due to a zinc boundhistidine and the appearance of signals representing zinc-free histidineindicates ejection of zinc (21). Preferably, said detection usesspectrofluorimetry as applied to the NCp7 zinc finger (22) with stopflow facilities used to measure on/off rates for zinc binding. Detectionof zinc using this method utilises several fluorescent indicators, whichexhibit an increase in fluorescence upon binding of zinc. Saidfluorescent indicators may, for example, be Newport Green™ orN-(6-methyoxy-8-quinolyl)-p-toluenesulphonamide (25). Thus, for example,said fluorescent indicators may be used to examine for zinc binding,strength of zinc binding and the effect of test agents on apolypeptide/zinc complex according to the present invention.

Generally speaking, any agents detected by the method according to thepresent invention that cause a change in said polypeptide/zinc complexmay be used as an antiherpesvirus agent.

In a further aspect, the present invention also relates to use of anIE63 polypeptide, functional derivative or functional homologue thereofor polypeptide comprising a zinc finger motif as defined herein in thedisclosed methods and use of a nucleic acid encoding said polypeptide,in the disclosed methods. Said polypeptide and its functional homologuesor derivatives thereof are as described earlier. Generally speaking, theskilled man will understand that the nucleic acid counterparts may beused for cloning, using standard techniques, nucleic acid constructsrequired for expression of said polypeptide or functional homologuethereof. Optionally, said nucleic acid may be used to clone theC-terminus encoding region of IE63 and its homologues, incorporating theconserved zinc finger motif according to the present invention.Furthermore, they may be used to express and purify said polypeptide. Amethod of purification of said polypeptide comprising a zinc fingermotif can utilise the motif itself. For example, the dimerisationproperties of a zinc finger motif can be utilised by binding saidpolypeptide to Glutathione beads, which are then used to purify furtherpolypeptide from herpesvirus-infected cell extracts throughself-interaction of the polypeptide. A further means of providingpolypeptide is use of automated peptide synthesis.

In a third aspect, the present invention provides agents detected by themethod according to the present invention and their use to treatherpesvirus infections. It should be understood that this may includeany suitable pharmaceutical composition comprising, as active substance,any said agent and any synthetic functional derivative thereof. Suchagents can also be used in conjunction with known antiherpesvirus agentsin the treatment of herpes viral infections as well as, if necessary,one or more pharmaceutical adjuvants. This composition may beadministered in any form by various means including topical applicationto the site of viral breakout eg. mouth for HSV-1 lytic cycle.

The discovery by the present inventors that the spacing and metalco-ordinating residues of the IE63 zinc finger and its homologues areconserved within each of the herpesvirus sub-families (alpha-, beta- andgammaherpesvirus; Example 1)is analogous to the retrovirus HIV-1 proteinNCp7, which has been targeted for the development of antivirals thatselectively eject zinc (reviewed in 2).

Thus, the present invention further provides the use of previouslyidentified antiretroviral agents in the treatment of herpesvirusinfection. Particularly, antiretroviral agents whose action is in theejection of zinc bound to a zinc finger protein, for example, C-nitrosocompounds such as 6-nitroso 1,2-benzopyrone (NOBP), 2-nitrosobenzamide,3-nitrosobenzamide (NOBA), 4-nitrosobenzamide,5-nitroso-1(2H)-isoquinolinone (5-NOQ), 7-nitroso-1(2H) isoquinolin(5-NOQ), 8-nitroso-1(2H)-isoquinolinone (8-NOQ), and related compoundsincluding nicotinamides, pthalhydrazides and 1,3-benzoxazine-2,4 diones;phenylthiols; dithiaheterocyclic molecules; disulphide benzamides orazoic compounds. Preferred antiretroviral agents for use according tothe present invention are the disulphide benzamide2,2′-dithiobisbenzamide (DIBA) and azoic compound, azodicarbonamide(ADA). Said antiretroviral agents were identified previously aseffective in the treatment of retroviruses only due to their action ofZn-ejection from the zinc finger motif of HIV-1 NCp7.

In a further aspect, the present invention provides the use of suchpreviously identified antiretroviral agents for the manufacture of amedicament for the treatment or prophylaxis of herpes virus infections.

In a yet further aspect there is provided a kit for diagnostic in vitrodetection of agents for use in the treatment of herpes virus infection,wherein the kit comprises:

-   a) a herpes virus polypeptide, said polypeptide comprising a    sequence selected from the group consisting of (at least) the    Cys-X₁₀-His-X₃-Cys-X₄-Cys-X₁₄-His-X₆-Cys alphaherpesvirus subfamily    zinc-finger motif, Cys-X₁₂-His-X₁-Cys-X₄-Cys-X₁₇-Cys betaherpesvirus    subfamily zinc-finger motif and His-X₃-Cys-X₄-Cys gammaherpesvirus    subfamily zinc finger motif of herpesvirus IE63 functional    homologues;-   b) a source of zinc, in order that when added to said polypeptide, a    polypeptide/zinc complex is formed; and-   c) means for enabling determination of any change in said    polypeptide/zinc complex in the presence of an agent.

It may be appreciated that the polypeptide and zinc may be provided as apreformed polypeptide/zinc complex.

Determination of agents for use in the treatment of herpes virusinfection is based on a change in the properties of saidpolypeptide/zinc complex when a test agent is added. Such a change maybe determined by methods using ⁶⁵Zn²⁺ or NMR, which can detect ejectionof zinc from said polypeptide/zinc complex by a test agent as describedearlier.

Typically, said polypeptide or polypeptide/zinc complex may beimmobilised on a solid substrate which may be, for example, a well of amicrotiter plate, cuvette, nitrocellulose or the like.

The present invention will now be further described by way of exampleonly, with reference to the following methods and figures which show:

FIG. 1—comparison of protein sequences of herpesvirus IE63 andhomologues. Alignment of amino acid sequences using the “Pileup”programme (Wisconsin Sequence Package, Version 9.0, Genetics computerGroup) of IE63 and its homologues from various human and animalherpesviruses including alpha-, beta- and gammaherpesvirus subfamiliesare shown.

FIG. 2—alignment of C-terminal region of IE63 and homoloques. Threealignments of the C-terminal region of IE63 and its homologues using theGCG “Pileup” programme are shown for alpha-, beta- and gammaherpesvirussubfamilies. The C-terminal region contains a conserved zinc fingermotif within each subfamily of herpesvirus. Conserved residues are shownin bold and denoted by the single letter conserved amino acid residueunderneath.

FIG. 3—Gel showing Purification of His tagged IE63 SDS PAGE gel showingthe fractions containing IE63 obtained by purification on Qiagen Ni-NTA.

FIG. 4—Western Blot of IE63 Purification Western Blot of fractionobtained from purification of IE63 on Ni-NTA.

FIG. 5—Zinc election from IE63 monitored by spectrofluorimetry Zincejection assay performed on HSV-1 IE63 protein as described in (24). 25μM TSQ and 0.5 μM IE63 added at time point X. 25 μM DIBA added at timepoint Y. Excitation wavelength 360 nm and emission wavelength 460 nm,sampling every 2 sec.

METHODS

Cytotoxicity. Both compounds azodicarbonamide (ADA, NSC 674447) and2,2′,-dithiobisbenzamide (DIBA, NSC 654077) were dissolved at theappropriate concentration in 0.1% (v/v) dimethylsulphoxide (DMSO) inEagle's modified medium. Confluent monolayers (2×10⁶) of baby hamsterkidney BHK21/C13 cells were incubated with various concentrations ofeither ADA or DIBA (1,10,100 μM) or as a control 0.1% (v/v) DMSO, andcell viability determined every day for 4 days by Trypan Blue exclusionstaining (23).

Virus replication. Semi-confluent monolayers of BHK21/C13 cells (2×10⁶)were pre-incubated with the appropriate drug concentration, 0.1% DMSO(v/v) or mock treated as a control for 24 h and subsequently infected ata multiplicity of infection (moi) of 5 plaque forming units (pfu)/cellwith either herpes simplex virus type 1 (HSV-1) strain 17 or HSV-2strain HG52 and harvested 24 h post-infection (pi). The virus yield wasdetermined by releasing the virus into the supernatant by sonication andtitration on BHK21/C13 cells. Results were expressed as pfu/10⁶ cellsand % reduction in yield as compared to the untreated control (23).

Plaque reduction assay. Confluent monolayers (4×10⁶ cells) of BHK21/C13cells were preincubated with the appropriate drug concentration, 0.1%DMSO(v/v) or mock treated as a control for 24 h and subsequentlyinfected at a moi of 400 pfu/plate with either HSV-1 or HSV-2 andincubated for 48 h at 37° C. The cells were fixed and stained withGiemsa stain and the number of plaques counted. Tests of each drugconcentration were performed in triplicate and the average of the 3plates calculated. Results were expressed as pfu/plate and % of thenumber of plaques on the untreated control plates (23).

Expression and Purification of HSV 1 IE63.

Expression of N terminal His Tagged IE63

E. coli Novablue cells (Novagen) were transformed with a pET28 vector(Novagen) containing the IE63 gene. Cells were plated on L-Broth Agarcontaining Tetracycline (12.5 μg/ml) and Kanamycin (25 μg/ml) andincubated overnight at 37° C. The following day a single colony wasinoculated into 10 ml of L-Broth containing tetracycline,chloramphenicol and 0.2% maltose and incubated at 37° C. overnight. Thenext day this 10 ml culture was inoculated into 500 ml of L-Broth(containing tetracycline, chloramphenicol and maltose) and grown at 37°C. until the OD₆₀₀ reached 1.0. MgSO₄ was added to a final concentrationof 10 mM and the λCE6 phage (Novagen) was added to a final concentrationof 2×10⁹ pfu/ml. The cells were grown for a further two hours and thenharvested by centrifugation. The pellet of frozen cells was storedfrozen at −20° C. until required. Protein expression was analysed byWestern blot using the IE63 antibody.

Purification of His Tagged IE63

The pellet of frozen cells was thawed slowly on ice and resuspended in 5ml of lysis buffer per 500 ml of culture.

-   Lysis Buffer-   2 mM Tris HCl pH 8-   100 mM NaCl-   0.5% NP-40-   20 mM imidazole

Protease inhibitor cocktail tablet (Boehringer). Cells were lysed byFrench pressing and the insoluble material pelleted by centrifugation at4° C. and 15000 rpm. The supernatant is removed to a clean tube and 1 mlof Ni-NTA (Qiagen) resin is added per litre of starting culture. Thismixture is then incubated with rotation at 4° C. for 2-3 hours. TheNi-NTA resin is pelleted by centrifugation at 3000 rpm for 1 min and thesupernatant removed. The resin is then washed with 2 times 50 ml oflysis buffer (described above) followed by one wash with lysis bufferminus NP-40, one wash with lysis buffer plus 1M NaCl and finally onewash with original lysis buffer. The washed beads are then loaded into a10 ml column (Biorad) and allowed to settle. The protein is eluted fromthe resin in four to five 1 ml aliquots of elution buffer

-   Elution buffer-   20 mM Tris HCl pH 8-   100 mM NaCl-   300 mM Imidazole

The fractions are then analysed by SDS PAGE and Western Blot for thepresence of IE63. Fractions containing IE63 are dialysed against 50 mMTris pH 8 containing 10% glycerol before storing at −20° C.

EXAMPLE 1 Conservation of Zinc Finger Regions in Herpesvirus IE63 andHomologues

Amino acid sequences of herpesvirus IE63 and its various human andanimal counterparts were compared using the GCG “Pileup” alignmentprogramme. It was observed that the spacing of the metal co-ordinatingcysteine and histidine residues in the IE63 zinc finger (FIG. 1) areprecisely conserved in all homologues within the alphaherpesvirussub-family (Cys-X₁₀-His-X₃-Cys-X₄-Cys-X₁₄-His-X₆-Cys) and for the othersub-families (FIG. 1) there is a similar conservation of spacing but thearrangements are different (betas; Cys-X₁₂-His-X₁-Cys-X₄-Cys-X₁₇-Cys,gammas; His-X₃-Cys-X₄-Cys). The zinc finger lies at the extremeC-terminus of IE63 and its alphaherpesvirus and betaherpesvirushomologues, and towards the C-terminus in the gammaherpesviruscounterparts. Inspection of the database reveals that these three zincfinger arrangements are different from those present in other identifiedviral and cellular proteins.

EXAMPLE 2

Determination or cytotoxicity of ADA and DIBA. In view of thedistinctive sequence conservation of the IE63 zinc finger two previouslyknown zinc-ejecting anti-HIV-1 compounds, 2,2′-dithiobisbenzamide (DIBA)and azodicarbonamide (ADA) were tested for their effect on herpesvirus.To separate any effect on cell viability from a specific effect on virusreplication, the effect of incubating BHK21/C13 cells in variousconcentrations of ADA, DIBA, and 0.1% (v/v) DMSO as a control, for up to4 days was determined. Even at the highest concentration used (100 μM)95% of the cells were viable compared to untreated BHK21/C13 cells (datanot shown). Indeed any cytotoxicity observed was due to the DMSO inwhich the drugs were dissolved, as the cytotoxicity was equally great inthose cells incubated with only DMSO (data not shown).

EXAMPLES 3

Antiviral effect of ADA and DIBA. Two experiments were carried out. Inthe first, the effect of varying concentrations of ADA and DIBA on thereplication of HSV-1 was analysed. In the second, the effect on bothHSV-1 and HSV-2 was determined. The results are summarised in Tables 1and 2 respectively.

TABLE 1 VIRUS YIELD VIRUS YIELD % UNTREATED (pfu/10⁶ BHK cells) (pfu/BHKcell) CONTROL HSV-1 0   8 × 10⁸* 800  100% 0.1% DMSO 7.2 × 10⁸ 720   90% 1 μM ADA 6.1 × 10⁸ 610   76%  10 μM ADA 1.5 × 10⁸ 150   19% 100 μM ADA  9 × 10⁶ 9.0   11%  1 μM DIBA 6.8 × 10⁸ 680   85%  10 μM DIBA 1.6 × 10⁸160   20% 100 μM DIBA   2 × 10⁷ 20  2.5% *each value represents anaverage of 2 plates

TABLE 2 VIRUS YIELD VIRUS YIELD % UNTREATED (pfu/10⁶ BHK cells) (pfu/BHKcell) CONTROL HSV-1 0   6.1 × 10⁸* 610  100% 0.1% DMSO   6 × 10⁸ 600  98%  1 μM ADA 9.3 × 10⁷ 93   15%  10 μM ADA 8.5 × 10⁶ 8.5  1.4% 100 μMADA 1.2 × 10⁶ 1.2 0.19%  1 μM DIBA 1.2 × 10⁸ 120   20%  10 μM DIBA 9.6 ×10⁶ 9.6  1.6% 100 μM DIBA 1.9 × 10⁶ 1.9 0.31% HSV-2 0   7 × 10⁷* 70 100% 0.1% DMSO 7.2 × 10⁷ 72  103%  1 μM ADA 9.4 × 10⁶ 9.4   13%  10 μMADA 8.5 × 10⁵ 0.85  1.2% 100 μM ADA 2.3 × 10⁵ 0.23 0.33%  1 μM DIBA 1.2× 10⁷ 12   17%  10 μM DIBA 1.4 × 10⁶ 1.4   2% 100 μM DIBA 3.1 × 10⁵ 0.310.44% *each value represents an average of 2 plates

With both compounds there was a dose dependent reduction in virus yield.In experiment 1, DIBA appeared to have a greater antiviral effect,reducing the virus yield to 2.5% compared to 11% for ADA at 100 μM.However, in the second experiment, where the overall antiviral effectwas greater, there was no significant difference between the effect ofDIBA and ADA, with virus yield being reduced to less than 1% of theuntreated control. Both HSV-1 and HSV-2 replication was equallyaffected.

EXAMPLE 4

Plaquing Efficiency in the Presence of ADA and DIBA. To determine if thereduction in virus yield seen in the previous experiment was due to avirucidal effect, inhibition of virus entry (adsorption penetration) orintracellular replication, a plaque reduction assay was carried out.Here, the ability of the virus to plaque was measured. An effect on thestability of the virus particle or virus entry would lead to a reductionin the number of plaques compared to the control untreated plates for agiven amount of virus. In contrast, an effect on intracellular virusreplication would not lead to a significant reduction in plaques. Nosignificant reduction in HSV-1 or HSV-2 plaquing was seen with at most a10% reduction (i.e. in the presence of the drug 90% of the number ofplaques seen with untreated plates still remained) seen at the highestdrug concentrations (data not shown). However, there was a reduction inthe size of the plaques consistent with an effect on virus replicationreducing the yield of progeny virus from each infected cell.

This experiment strongly suggests that the antiviral effect of DIBA seenin the previous experiment is due to an inhibition of intracellularreplication.

EXAMPLE 5

Zinc Ejection Assay

Purified HSV-1 IE63 protein was prepared as described earlier for use inthe zinc ejection assay. The zinc ejection assay for IE63 was carriedout as described in (24) by substituting the IE63 protein for the p7NCprotein. All chemicals were used at the same concentrations asdescribed. Results, as measured by spectrofluorimetry are shown in FIG.5.

It is understood that the above embodiments are merely representative ofthe present invention and should not be considered to be limitingthereof.

REFERENCES

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1. A method for detecting an agent for use in the treatment of herpesvirus infection comprising the steps of: (a) forming a herpes viruspolypeptidelzinc complex; (b) adding a test agent to saidpolypeptide/zinc complex; and (c) detecting any change in thepolypeptide/zinc complex, wherein the herpes virus polypeptide comprisesat least one zinc finger motif, and wherein detection of said agent isbased on a change in the properties of said polypeptide/zinc complex. 2.A method for detecting an agent for use in the treatment of herpes virusinfection according to claim 1 wherein the agent is selected from thegroup consisting of a chemical, nucleic acid analogue, peptide andprotein.
 3. A method for detecting an agent for use in the treatment ofherpes virus infection according to claim 1 wherein the agent is aC-nitroso and related compound and is selected from the group consistingof 6-nitroso 1,2-benzopyrone (NOBP), 2-nitrosobenzamide,3-nitrosobenzamide (NOBA), 4-nitrosobenzamide,5-nitroso-1(2H)-isoquinolinone (5-NOQ), 7-nitroso-1(2H) isoquinolin(5-NOQ), 8-nitroso-1(2H)-isoquinolinone (8-NOQ), and related compoundsincluding nicotinamides, pthalhydrazides and 1,3-benzoxazine-2,4 diones;phenyithiols; dithiaheterocyclic molecules; disulphide benzamides andazoic compounds.
 4. A method for detecting an agent for use in thetreatment of herpes virus infection according to claim 1 wherein theagent is 2,2′-dithiobisbenzamide (DIBA).
 5. A method for detecting anagent for use in the treatment of herpes virus infection according toclaim 1 wherein the agent is azodicarbonamide (ADA).
 6. A method fordetecting an agent for use in the treatment of herpes virus infectionaccording to claim 1 wherein the polypeptide is selected from the groupconsisting of herpes virus HSV-1 and HSV-2 IE63 protein.
 7. A method fordetecting an agent for use in the treatment of herpes virus infectionaccording to claim 1 wherein the polypeptide is selected from the groupconsisting of VZV ORF4, HCMV UL69, Epstein-Barr virus BMLF1 (SM/MTA),HHV-6 U42, HHV-7 U42, HHV-8 ORF57, equine herpes virus-1 ORF3, equineherpes virus-4 ORF3, bovine herpes virus-1 BICP27, pseudorabies virusUL54, Marek's disease virus UL54, murine cytomegalovirus M69, herpesvirus saimiri ORF57, bovine herpes virus 4 ORF57, avian herpes virus-1ORF57 and murine herpes virus-68 ORF57.
 8. A method for detecting anagent for use in the treatment of herpes virus infection according toclaim 6 wherein the polypeptide comprises at least the C-terminalregions of IE63.
 9. A method for detecting an agent for use in thetreatment of herpes virus infection according to claim 1 wherein thepolypeptide comprises zinc finger motifs selected from the groupconsisting of Cys-X₁₀-His-X₃-Cys-X₄-Cys-X₁₄-His-X₆-Cys alphaherpesvirussubfamily zinc-finger motif, Cys-X₁₂-His-X₁-Cys-X₄-Cys-X₁₇-Cysbetaherpesvirus subfamily zinc-finger motif and His-X₃-Cys-X₄-Cysgammaherpesvirus subfamily zinc finger motif.
 10. A method for detectingan agent for use in the treatment of herpes virus infection according toclaim 1 wherein said zinc is provided in the form of a standard dialysisbuffer comprising a known concentration of zinc.
 11. A method fordetecting an agent for use in the treatment of herpes virus infectionaccording to claim 1 wherein said change in the properties of saidpolypeptide/zinc complex is measured by standard amino acid analysis andatomic absorption spectroscopy.
 12. A method for detecting an agent foruse in the treatment of herpes virus infection according to claim 1wherein detection of said agent is a measure of the test agent's abilityto eject zinc and/or destabilize viral zinc fingers.
 13. A method fordetecting an agent for use in the treatment of herpes virus infectionaccording to claim 1 wherein detection of said change in the propertiesof said polypeptide/zinc complex is achieved by using a selection fromthe group consisting of ⁶⁵Zn²⁺ and NMR.
 14. A method for detecting anagent for use in the treatment of herpes virus infection according toclaim 1 wherein detection of said change in the properties of saidpolypeptide/zinc complex is achieved by using spectrofluorimetry withstop flow facilities.
 15. A method for detecting an agent for use in thetreatment of herpes virus infection according to claim 1 whereindetection of said change in the properties of said polypeptide/zinccomplex is achieved by using spectrofluorimetry with stop flowfacilities and wherein said spectrofluorimetry utilizes severalfluorescent indicators, which exhibit an increase in fluorescence uponbinding of zinc.
 16. A method for detecting an agent for use in thetreatment of herpes virus infection according to claim 15 wherein saidfluorescent indicators include Newport Green™ orN-(6-methyoxy-8-quinolyl)-p-toluenesulphonamide.
 17. A method fordetecting an agent for use in the treatment of herpes virus infectionaccording to claim 15 wherein said fluorescent indicators are used toexamine zinc binding, strength of zinc binding and the effect of saidtest agents on said polypeptide/zinc complex.